Project Summary The long-term objective of this project is to develop a new class of broad spectrum antibiotics, focused on Gram-negative bacteria and tuberculosis. In addition, the proposed project will teach how to optimize inhibitors of the prokaryotic ribosome by defining critical interactions only possible in bacteria. Inspired by a natural product, this class targets an unexploited binding site of the bacterial ribosome. With increasing reports of resistance to frontline antibacterial therapies, there is a critical need for new agents, yet very little can be found in the development pipeline. As a result, any new therapeutic that targets Gram-negative bacteria and/or tuberculosis will address an unmet medical need. A viable approach to discover new therapeutic leads is re-evaluation of existing, but under-scrutinized classes of natural products. Through this approach a natural product scaffold was identified as a lead for a program directed toward antibiotics for tuberculosis. Initial evaluation of activity against other bacteria indicated that the antibacterial spectrum was limited to Mycobacterium spp. However, based on the chemical structure and related compounds, we expected to be able to generate analogs with more broad spectrum activity. In collaboration with Tom Steitz's lab at Yale, the structure of the initial scaffold and two analogues bound to the ribosome were recently solved. The structural studies revealed that the natural product lead and analogues bind to a highly conserved region of the peptidyl transferase center (PTC) in a manner that appears to convey prokaryotic selectivity and which has not been exploited in current therapeutics. Targeting this highly conserved region is expected to lead to slow rates of resistance. This structural information was used to generate two more potent analogues with favorable physiochemical properties. In a very preliminary SAR campaign of ~30 compounds, we re-engineered a portion of our lead scaffold for both synthetic simplicity and stability to provide two analogs that introduce activity beyond Mtb, to MRSA, E. coli and K. pneumoniae (including a carbapenem-resistant strain), and do not exhibit cytotoxicity to eukaryotic cells (IC50 >100 M). This project will explore and further define critical binding interactions for inhibitors to the bacterial ribosome that impart selectivity for inhibiting bacterial protein synthesis and broad spectrum antibacterial activity, while maintaining the lack of mammalian cytotoxicity. Aim 1 will expound on our hypothesis for how compounds can selectively bind to prokaryotic ribosomes at an undrugged site. If our hypothesis is correct, these inhibitors will not be active against mammalian ribosomes, thereby mitigating limitations of other ribosome-targeting antibiotics. Aims 2 and 3 will explore features of related compound families that, based on our hypotheses, are expected to facilitate more potent inhibition of bacterial ribosomes while maintaining selectivity/safety and enhancing broad spectrum antibacterial activity.